Recently, organ transplantation has been widely carried out in various countries. Development of highly effective immunosuppressants (Cyclosporin, FK506 and the like) has solved the problems of rejection of organs transplanted from man to man, however, lack of donors has become a serious problem. Such a problem has prompted studies on animal-to-man organ transplantation, namely xenotransplantation. Although approximately 3,500 heart transplantations are being performed annually in European countries and the United States, they cover only approximately 20 to 30% of patients who need heart transplantation. Use of animals closely related to human beings as donors (for example, such primates as baboons, chimpanzees and the like) involves a great deal of difficulty due to shortage of these animals and their high intelligence, but use of domestic animals as donors involves less problems. Particularly, pigs have advantages of easy supply due to mass rearing, their organ sizes similar to those of man, and established basic technology including maintenance of the strains. Consequently, organ transplantation from pigs to man has been studied.
Of rejections occurring in pig-to-man organ transplantation, acute rejection by Major Histocompatibility Complex (MHC)-related cellular immunity may not occur, since evolutional relatedness between pigs and man is so scarce that there is no similarity between their MHCs. Moreover, application of such effective immunosuppressants may avoid such rejection, if ever occurs.
Human blood, however, contains endogenous antibodies against pigs (namely, natural antibodies). Consequently, if a porcine organ is transplanted to man, the natural antibodies recognize the organ (antigen) resulting in formation of antigen-antibody complexes, which activate human complements. The activated human complements cause necrosis of the transplanted organ (rejection). Such a phenomenon occurs immediately (within an hour) after transplantation, so it is termed hyperacute rejection.
No drug inhibiting hyperacute rejection caused by complement activation has ever been developed. No human organ is injured by human complements, since factors preventing complement activation are expressed in human organs. Such factors are named complement inhibitors (or complement-inhibiting factors). Of the complement inhibitors, three factors, DAF (decay accelerating factor, CD55), MCP (membrane cofactor protein, CD46) and CD59, are important. It is believed that DAF and MCP inhibit activation of complements by accelerating the destruction of C3b and C3/C5 convertase, and CD59 does so by inhibiting the C9 step.
The complement inhibitors are species-specific. Porcine complement inhibitors can inhibit the complement activity of pigs but not that of man. The porcine complement inhibitors cannot inhibit human complements activated by the porcine organ transplanted to man. Therefore,the porcine organ transplanted to human undergoes necrosis.
Such problems arising when a porcine organ is transplanted to man will be solved, if human complement inhibitors are expressed in the porcine organs by genetic engineering. In transplantation of the porcine heart, there will be no problem if the human complement inhibitors are being expressed by porcine vascular endotherial cells. From such a viewpoint, studies on recombinant pigs (transgenic pigs) integrated with human complement-inhibitor genes have widely been carried out.
As described above, xenotransplantation by using transgenic pigs integrated with the human complement-inhibitor genes have been studied. Up to the present, promoters derived from the human complement-inhibitor gene and from viruses have been used to prepare such transgenic pigs. For the complement inhibitors to be expressed in pigs, however, the promoters orginating from pigs may be more efficient. To obtain such promoters, cDNA of the porcine complement inhibitors is needed, however, no such cDNA has ever been known.
From these viewpoints, the present inventors conducted cloning for expression of porcine complement-inhibitor cDNA and succeeded in obtaining cDNA which could make the cells resistant against the porcine complement and highly homologous to human MCP. During the course of these studies, the inventors found also a novel method for screening for cDNA libraries.
This invention was accomplished on the basis of such findings. The purposes of the invention were to provide cDNA for the porcine complement inhibitor, the porcine complement-inhibitor protein, and the method for screening for the complement-inhibitor gene.